Unusually isolated Staphylococcus arlettae in intra-oral sutures - Case series

Introduction. The human oral cavity comprises various niches such as teeth, gingiva, tongue, soft and hard palate, and various dental prostheses, all inhabited by different bacterial species. Although more than 600 taxa belong to the oral cavity, identifying Staphylococcus arlettae , an incompletely understood bacterium, has been rare. Methods. Three patients who underwent periodontal flap surgeries were reported with the incidental finding of S. arlettae associated with the intra-oral sutures placed. Environmental sampling was performed, to establish the exact source of this bacterium. Results. Staphylococcus arlettae was isolated in three patients’ intra-oral sutures. All environmental samples were negative for the presence of the bacterium. Conclusion . To this date, no studies have identified such an occurrence of Staphylococcus arlettae with intra-oral sutures. Its identification in association with foreign materials, such as sutures, can be considered a potential for surgical site infections and requires further investigation.


INTRODUCTION
Previously, the coagulase-negative Staphylococci were thought to be non-pathogenic since they lacked the ability to clot blood plasma.They are, however, among the most prevalent organisms to cause bacteremia associated with indwelling devices worldwide.Staphylococcus arlettae, first isolated from farm animals' skin and respiratory organs, is a Gram-positive coagulasenegative coccus, non-motile, non-sporulating, occurring singly, in pairs, groups, or short chains [1].These are facultative anaerobic bacteria and are known to grow exuberantly on Brain Heart Infusion Agar (BHIA) with colonies appearing opaque, yellowish white or beige coloured, with complete margins, 6 to 8 mm in diameter after 2 days of incubation [1].Although the pathogenicity of this bacterium is not very clearly understood; it has been attributed to the polysaccharide components enabling persistent attachment to foreign materials.Most coagulase-negative organisms are hospital-acquired making them the frontiers of nosocomial infection, adding to morbidity and excess medical costs.Isolation of the S. arlettae in the clinical samples has been a rare occurrence, with only a few reports identifying a particular strain in the blood of cardiovascular disease patients [2].In contrast, others describe their incidence on cell phone surfaces and disused laboratory units [2][3][4].Here, we have isolated S. arlettae from the intra-oral sutures placed in three patients who underwent treatment with open flap debridement for chronic periodontitis.

CASE REPORT
Three patients, a 61 year-old-male, 41 year-old-female and 24 year old female, presenting to the Department of Dentistry were diagnosed with chronic periodontitis [5].The persistence of periodontal pockets warranted surgical therapy wherein open flap debridement by raising a Kirkland flap was done for all four quadrants of the oral cavity in a staged approach following the sterile surgical protocol [6].As the patients were enrolled in an ongoing study for microbiological assessment of the suture sample, the suture on removal was sent for microbiological culture.Those with reported identification of Staphylococcus arlettae from their suture samples were included for further study.Since the occurrence of samples positive with Staphylococcus arlettae culture was an event by chance, no randomization, blinding or power analysis of sample size was done.All three patients were requested to follow up at 1 month from the time of identification of Staphylococcus arlettae in the sample for any signs of fever, pain, or delayed wound healing and to report the incidence of any such untoward experience post-operatively.Wound healing was assessed at the time of suture removal and 1 month post-operatively, using the Early Wound Healing Score [7] (Fig. 1a).

BACTERIAL CULTURE OF SUTURE SAMPLES
The intra-orally placed sutures (MITSU polyglactin 910 sutures, MERIL Life Sciences, Pvt, Ltd) for periodontal flap approximation were aseptically removed on eighth post-surgical day and collected in 5 ml Thioglycolate Broth (HiMedia, Mumbai, India) in sterile tubes.These were vortexed for 2 min.After a serial dilution up to 1 : 10 6 , 0.1 ml aliquot of the final diluted sample was then cultured on Blood Agar (HiMedia, Mumbai, India) and incubated at 37 °C for 24 h.Individual colonies were then sub-cultured and processed immediately as per routine hospital procedure of examination, including bacterial identification, Gram-staining (HiMedia, K001, Mumbai, India), colony morphology characterization by size, shape, texture, opacity and identified by the VITEK two automated identification system, (bioMérieux, Marcy, France).A confirmatory identification using the MALDI-TOF assay (MALDI Biotyper, Bruker) was done for isolated colonies of Staphylococcus arlettae.Isolated colonies were also subjected to antibiotic susceptibility assays which were performed using an automated system -VITEK 2 AST cards (bioMérieux, Marcy, France) following the guidelines from Clinical and Laboratory Standards Institute (CLSI), USA [8].Demographical distribution along with antibiotic susceptibility pattern of three cases included in the present study have been mentioned in the Table 1.

ENVIRONMENTAL SAMPLING
In order to eliminate the possibility of bacterial contamination from the environment at any of the several steps in sample processing, samples representing the environment were taken for isolation of S. arlettae.Samples representing the dental chair environment and inoculation environment in the microbiology laboratory were taken by the settle plate method [9].Water from dental water unit was inoculated in blood and nutrient agar plates.These plates were then incubated at 37 °C for 24 h and observed for growth.The thioglycolate broth bottles and sterile saline bottles for dilution were compared for optical density before and after incubation at 37 °C for 24 h to check for any contaminant within [10].Sterile sutures from the same batch of manufacturing as those used for the surgeries were inoculated in 5 ml of nutrient broth and observed for growth after incubation at 37 °C for 24 h (Fig. 1b, c).

BACTERIAL CULTURE OF PATIENT PLAQUE SAMPLE
Supra-gingival plaque was collected from the patients with reported incidental findings of S. arlettae at their subsequent visit to the OPD (about 1 week) using sterile curettes and placed in a sterile bottle with 5 ml thioglycolate broth.The bottle was vortexed for 2 min, followed by inoculation of 0.1 ml of the solution on blood agar and nutrient agar plates by intermittent heating and streak method.After incubation, the culture plates were observed for growth.Based on colony morphology, colonies similar to that of S. arlettae were further sub-cultured and subjected to identification by the VITEK two automated identification system, (bioMérieux, Marcy, France).

SEM OF SUTURES -IN VITRO BIOFILM PRODUCTION
An in vitro biofilm production was carried out by placing 1-inch-long sterile suture samples in pure culture of S. arlettae and then subjected to scanning electron microscopy (SEM) to get detailed morphological evidence at submicron level.Briefly, isolated sutures were collected and immediately fixed with a phosphate buffer saline solution containing 2 % (vol/ vol) formaldehyde for 24 h.Fixed sutures were gradually dehydrated using a series of ethanol gradients (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%).Sutures were fixed for 20 min at each ethanol gradient.Next, sutures were sputter coated with a layer of gold and observed under scanning electron microscope (Aprio, Thermo Fisher Scientific, USA) (Fig. 2).

RESULTS
Samples of the three patients yielded an incidental identification of S. arlettae from cultured suture samples at different time intervals.All of the environmental samples collected were negative on culture for S. arlettae.Furthermore, we performed antibiotic susceptibility testing, and the results are shown in Table 1.Dental plaque samples collected from the same site as the prior suture failed to show the presence of S. arlettae after approximately 1 week of suture removal.Other organisms identified  included Staphylococcus gallinarum, which is very similar to S. arlettae, except that they are positive for cellobiose and urease reactions [1].One of the patients reported gnawing pain and discomfort up to 4 days post-operatively.On examination and evaluation of the healing of the surgical site, no apparent inflammation or deviation from standard healing patterns was present.On 1 month follow-up, these patients showed satisfactory healing, with no reported fever, wound infection or pain during the healing period.Oral hygiene status was good for all three patients.Finally, our SEM results showed high biofilm formation with both types of sutures as compared to the control.

DISCUSSION
It is apparent that S. arlettae has a predilection for foreign material surfaces, even though the specific virulence factors of the bacterium are not as well established as they are in Staphylococcus aureus [11].The literature for its occurrence in human samples is sparse, from being considered as normal skin commensals to being associated with different types of infections especially where a large number of antibiotics have been used [2,3,[12][13][14].This makes its role in surgical site infections questionable.It has been considered as an emerging opportunistic pathogen with an antibiotic resistance profile similar to that of the prominently pathogenic Staphylococcus species -S.aureus and Staphylococcus epidermidis and virulence factors similar to Staphylococcus hemolyticus and Staphylococcus saprophyticus [4].In the past few decades, coagulase-negative Staphylococci have been recognized as one of the important nosocomial pathogens and they also ranked among the top two most frequently isolated organisms in hospital wards and ICUs from the central line associated-bloodstream infections [15].
To the best of our knowledge, this is the first incidence of S. arlettae to be isolated from intra-oral samples associated with sutures.Previously, in a multi-centric study which determined the contamination of water in dental unit reservoirs from 107 dental unit reservoir samples located in dental surgeries of public health centres, S. arlettae was reported with an incidence rate of 1 (0.93 %) [16].However, in our setting, we failed to identify any bacterial growth associated with the water samples taken from the dental water unit lines.Clinically, none of the patients could be identified with any significant morbidity attributable to the presence of this bacterium.Whether this bacterium is associated with disease or worsening of periodontal state is still a missing piece of the puzzle and requires further studies.However, by understanding the microbiological characteristics of S. arlettae, we can opinionate on its occurrence in the oral cavity in association with intra-oral sutures as follows: Virulence factors of this species include specific bacterial binding to extracellular matrix molecules such as laminin, fibronectin, vitronectin, fibrinogen, collagen and others by means of various cell wall proteins [17].Microscopically identified fimbriae-like structure on this bacterium by electron-microscopy may also be considered as an important role in attachment to foreign materials in the host [18].Isolating S. arlettae from intra-oral sutures and its absence in subsequent plaque samples or environment points to an important clue that it is probably present as a commensal in detectably low numbers, kept in check by the host's immunity under normal conditions.In the presence of any foreign implantable material, it may exhibit exuberant growth and form a biofilm.The tendency of S. arlettae to be associated with foreign materials may result in an increased probability of surgical site infections, causing morbidity to the patient.In our study, one of the three patients reported of persistent gnawing pain at the surgical site and discomfort post-operatively.Pain is however a subjective parameter with varying degrees of assessment and its causative factors can be many, such as surgical manipulation, post-operative care, and therefore difficult to be identified.Also, healing was assessed only clinically, which overlooks the possibility of altered healing at histologic levels.
In our study, the suture material was removed at eighth day post-operatively.However, the resorbable nature of the suture might be convincing to certain clinicians and patients alike to let the suture stay to resorb on its own for longer periods of time.This, however may be disastrous in a situation where such non-coagulase-positive Staphylococci are adherent onto the suture material, allowing its port of entry into the adjacent tissues, especially in an added disadvantage of immune-compromised state of the host.This could be worsened by the presence of multi-drug resistant strains of this species.However, further studies are required to prove the pathogenicity of this bacterium in regard to the oral environment.
Another important point to be stressed is the use of chemical plaque control post-operatively in patients undergoing periodontal flap surgery.The compromised mechanical plaque control that follows the periodontal surgery can increase the plaque formation on the suture materials.An antimicrobial oral rinse such as 0.2 % chlorhexidine digluconate remarkably reduces the bacterial load on the suture materials [19].Studies have shown that chlorhexidine is effective in reducing coagulase-negative bacterial counts by up to 63.2 % and, thus, should be routinely prescribed to keep pathogenic bacterial counts under control [20].Chlorhexidine coated sutures with increased drug release locally can add to the benefit of the chemical plaque control and is evident from the SEM results of our study as lesser biofilm formation occurred with the use of coated sutures.

CONCLUSION
Coagulase-negative Staphylococci are emerging as opportunistic pathogens and their occurrence on intra-orally placed sutures is an alarming sign, considering the possibility of bacteremia.Duration of sutures in situ for wound approximation should be as brief as possible and plaque control in the post-surgical healing phase should be emphasized on.We also urge readers to avoid dismissing the atypically cultured bacteria as commensals or contaminants too easily and encourage discussion to improve our understanding of the microbiological world.

VERSION 2
Editor recommendation and comments https://doi.org/10.1099/acmi.0.000555.v2.1 © 2023 Rudkin J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.
Justine Rudkin; University of Oxford, Nuffield Department of Population Health, UNITED KINGDOM, Oxford Date report received: 05 July 2023 Recommendation: Minor Amendment Comments: Thank you for addressing some of the reviewers comments.The manuscript is in a good state and is suitable for publication as it is, but I would like some minor edits to be done before sending it through for final publication please.1. Line 39-please add "however" in between They are *however* among the most..... 2. Line 52/53-the authors state "Here, we have isolated a S. arlettae strain", but you have isolated multiple strains-the AMR data indicate that the isolations from each patient are not the one strain.Please amend the sentence.3. Line 181-I don't think the manuscript referenced in ref No.18 can really be described as a recent observation of fimbriae-like structures-it was published in 1996.Please amend the sentence.4. Lines 200-202-it is still not clear what role genome sequencing would play in "prove the pathogenicity of this bacterium in regard to the oral environment".I would change the sentence to say "However, further studies are required to demonstrate the pathogenic potential of this bacterium in regard to the oral environment."Other than those minor things, it is good to go.

RESPONSE TO REVIEWERS
Respected Reviewers, We appreciate and thank you for your precious time in reviewing our paper and providing valuable comments.They were indeed very insightful which has led to possible improvements in the current version.The authors have carefully considered and pondered over every suggestion and jointly addressed each of them as follows:

Sr no.
Comments Changes

1.
Line 39-40: "However, currently, they are among…" "however" or "currently", not both Comments: [Delete this text before submitting your review.Please include comments to the author here, and include the below sections, where possible.All comments here will be posted publicly online alongside the article once the Editor has made a decision.]1. Description of the case(s) The article titled "Unusually isolated Staphylococcus arlettaein intra-oral sutures-Case series" describes the isolation and identification of a coagulase negative Staphylococcal species in clinical samples.S. arlettaeis rarely found in a clinical setting and has not been studied to the same degree as similar Staphylococcal species, so less is known about its environmental preferances.The study gives a succinct methodology and identification, as well as discussion about where the organism may be located in the clinical setting.2. Presentation of results The results followed the methods and were succinctly presented.3. How the style and organization of the paper communicates and represents key findings The style and organization of the paper were done well and communicated to the audience what they needed to know about the results.4. Literature analysis or discussion The literature analysis was mostly good.It would help the authors case to cite a reference(s) about S. arlettaebeing teh cause of disease.The Discussion starts with talking about the virulence factors that S. arlettaedoes and doesn't possess, but then goes on to debate whether it is a commensal or not.Being associated with a disease in one immunocompromised individual is not the same as being consider virulent, at best it is an opportunistic pathogen.The same case could be made for S. epidermidis, that is is a commensal that can be an opportunistic pathogen, and that the organism is able to cause disease by exploiting innate physiology and not virulence factors that are specific to a host.Line 207-209: What benefit would whole genome sequencing do to prove pathogenicity if there is no normal disease state?The final paragraph of the discussion seems out of place in recommending a remedy when no further studies were performed to show that burden of S. arlettaein sutures is reduced when treated with Chlorhexidine.In particular, you just spent time making an argument that S. arlettaeseems to have a preferred environmental niche that may indicate it would be harder to remove from man-made substances like sutures.In the conclusion, one again, S.
arlettaewere not shown to be the sole cause of bacteremia. 5. Any other relevant comments My recommendation is to accept with minor revisions.There are a few concerning items in the discussion and conclusions, but are minor.In addition, the manuscript would benefit from significant proof-reading for grammatical errors.The items elow need attention: Line 39-40: "However, currently, they are among…" "however" or "currently", not both Line 52: It would be good to cite a case of S. arlettaein blood of cardiovasular disease patient Line 127 & 128: "in vitro" should be italicized Fig2: Caption for part "D" is missing.Line 168: Should be "epidermidis" not "epidermis" Line 186: Formatting-double comma Line 187: sp.Fibriae-like Line 221: "in situ" should be italicized Table 1-Last row: italicize bacteria names Access Microbiology uses Vancouver citation style, it would make the paper easier to review if this was done ahead of time.

Please rate the quality of the presentation and structure of the manuscript Good
To what extent are the conclusions supported by the data?Partially support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes

Fig. 1 .
Fig. 1. Materials and methods: (a) Clinical presentation of three cases at Day 0 (day of surgery), Day 8 (suture removal) and Day 30 (follow-up) assessing the wound healing.(b) Staphylococcus arlettae solation in pure form on blood agar and BHIA (brain heart infusion agar) media, and bacterial morphology by SEM.(c) Environmental sampling on NAM (nutrient agar medium) and blood agar from dental chair -showing β hemolytic organism growth and dental water unit lines -showing no microbial growth, microbiology laboratory workstation (BLST) (biosafety cabinet type-IIA) -showing growth other than S. arlettae and sterile suture on incubation -showing no microbial growth).

Table 1 .
Summary of data.Demographical distribution along with antibiotic susceptibility pattern of three cases included in the present study

Table 1 -
Last row: italicize bacteria names ItalicizedIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes